Our primary goal is the development, testing and eventual production of a novel class of anti-HIV therapeutics based on a general strategy referred to as Capsid-Targeted Viral Inactivation or CTVI. The core of the CTVI strategy is the intracellular production of antiviral fusion proteins consisting of a virus structural protein fused to a nuclease or other destructive protein; these fusion proteins are targeted to assembling virions. We will introduce genes encoding HIV capsid protein- nuclease fusion proteins into human cells and monitor their incorporation into HIV particles and their ability to inactivate virus particles enzymatically. For CTVI to be effective against HIV, the assembling inside these T cells because it is fused to HIV Gag or Gag/Pol protein. HIV capsid proteins self-assemble around the viral RNA genome. The infectivity of the hybrid HIV particle that carries the interfering fusion protein will be neutralized when the nuclease cleaves the viral genome. The first of two major goals of this project is to design and construct a set of HIV-Gag-nuclease fusion proteins and characterize and test them for antiviral efficacy. The second major goal will be to develop efficient and effective gene therapy vectors to deliver such antiviral constructs to human cells, and eventually, into animal models for AIDS, and ultimately into HIV-vector. AAV has many advantages over conventional retrovirus vectors, particularly for gene therapy applications, such as site-specific integration, integration into non- dividing cells, stable virus particles, and lack of pathogenic effects in humans. In this project, the antiviral approach will be tested independently against HIV-1 and SIV. In this way materials will be generated for eventual primate studies as well as for human clinical testing, although primate and human studies are beyond the scope of this grant application and are not included here.